The neurotransmitter, dopamine, modulates excitatory and inhibitory neurotransmission in brain regions that control movement, emotion, and reward. Dysregulation of DA transmission is implicated in the etiology of several pathological conditions including Parkinsons disease, drug addiction, and schizophrenia. The dopamine transporter (DAT) is an integral membrane protein that is a member of the Na+- and Cl-- dependent cotransporter gene family. It serves a key role in terminating dopamine transmission by clearing dopamine released into the extracellular space. Systemic administration of the endocannabinoid, anandamide, increases extracellular dopamine concentrations in the nucleus accumbens, a brain region implicated in mediating the abuse liability of various psychoactive drugs. Increased dopamine concentrations are also observed in response to synthetic cannabinoid agonists. Although these effects have been attributed to alterations in release, evidence that anandamide and other cannabinoids inhibit transporter-mediated dopamine uptake in native tissue and heterologous expression systems has been presented. Such findings are noteworthy in that they suggest that endocannabinoids may modulate dopamine transmission by regulating DAT function. The cellular mechanisms mediating the interaction of anandamide with DAT are unclear. Although there is evidence that it may modulate DAT function by a CB1 independent mechanism, whether the effect of anandamide is mediated by the activation of other cannabinoid receptors such as CB2 and GPR55 that are expressed in the brain is unclear. In addition, although prolonged incubation of cells or synaptosomes decreases dopamine uptake, the time course and the cellular mechanisms of this effect have not been assessed. We previously demonstrated that use of the fluorescent, high affinity DAT substrate, ASP+ 4-(4-(dimethylamino)styrl)-N-methylpyridinium))in combination with confocal microscopy enables real-time, spatially resolved analysis of transporter function in heterologous expression systems. Analysis of ASP+ accumulation not only permits resolution of substrate binding and uptake in the same cell but enables quantification of rapid (e.g. 1 min) changes in DAT function (see: Bolan et al., 2007, Zapata et al 2007). Using this technique we have investigated the effect of anandamide on the function of human DAT (hDAT) expressed in the EM4 HEK 293 cell line which does not endogenously express known cannabinoid receptors. In parallel studies, biochemical and confocal imaging techniques were employed to assess the role of transporter trafficking in mediating anandamide-evoked alterations in DAT function. Addition of anandamide to EM4 cells transiently transfected to express fluorescently tagged hDAT produced a concentration-dependent inhibition of ASP+ accumulation. This effect was rapid occurring within 1 min after anandomide addition. Increased DAT function was long-lasting. A significant inhihibition of ASP+ accumulation was still apparent 10 min after anandamide addition. Incubation of cells with pertussis toxin did not attenuate the effects of anandamide suggesting a mechanism independent of Gi/Go coupled receptors. The amidohydrolase inhibitor, phenylmethylsulfonyl fluoride, failed to alter the effects of anandomide. No change in ASP+ accumulation was observed in response to arachidonic acid suggesting that the effects of anandamide are not mediated by its metabolic products. The downregulation of DAT function was associated with a significant redistribution of hDAT from the membrane to the cytosol as measured using both confocal microscopy and biotinylation techniques. These results demonstrate that anandamide modulates DAT function via a cannabinoid receptor-independent mechanism. Furthermore, they suggest that endocannabinoids may increase extracellular dopamine, in part, by increasing DAT internalization.